Toxoplasma gondii, a protozoan parasite, is the causative agent of toxoplasmosis. Although benign in normal individuals, it is a serious problem for immune compromised hosts, such as patients with acquired immune deficiency syndrome (AIDS), and can also cause fetal damage if infection occurs during pregnancy.
The current treatment of choice for toxoplasmosis is the synergistic combination of pyrimethamine with sulfonamides. The toxicity associated with these drugs produces a significant treatment failure in AIDS patients and precludes its use during pregnancy. The motivation to find alternative drugs to combat this parasite is high. ##STR1##
Qinghaosu (QHS) is a sequiterpene lactone natural product derived from the Chinese herb Artemisia annua (I, where X=O). Artemisia annua has been used for centuries in China as a treatment for fever and malaria (Klayman (1985) Science 228:1049-1055). QHS (also called artemisinine, arteannuin, or artemisinin) has been shown to have in vitro and in vivo activity against Plasmodium (Qinghaosu Antimalaria Coordinating Research Group (1979) Chinese Medical Journal 92:811-816; China Cooperative Research Group on Qinghaosu and Its Derivatives as Antimalarials (1982) Journal of Traditional Chinese Medicine 2:17-24; Jiang et al. (1982) Lancet ii:285; Li et al. (1983) Transactions of the Royal Society of Tropical Medicine and Hygiene 77:522-523; Klayman et al. (1984) Journal of Natural Products 47:715-717).
Although QHS has been shown to have antimalarial activity, it is sparingly soluble in water or oils and is not well absorbed by the gastrointestinal tract. Derivatives with better bioavailability were sought. Dihydroqinghaosu (DHQHS) (I, where X=OH) is the product of reduction of the lactone group of QHS to a lactol (hemiacetal) which is reported to be a mixture of .alpha.- and .beta.-epimers. DHQHS is reported to be more effective than QHS against malaria. DHQHS derivatives, substitutions at the OH of DHQHS, have been found to be effective against malaria including alkyl ethers (I, X=OR, where R=alkyl, esters R=CO--alkyl or CO--aryl and carbonates R=CO--O--alkyl or aryl). Additionally, half esters of diacids such as succinate have been found to be effective against malaria. Such esters are prepared as salts i.e., sodium salts. A review of reports of methods used to prepare these derivatives from the natural product QHS is provided in Klayman (1985) Science 228:1049-1055. Artemether is used to refer specifically to the .beta.-epimer of dihydroqinghaosu methyl ether. Similarly the name arteether is used to refer to the .beta.-epimer of dihydroqinghaosu ethyl ether. Artesunate, in contrast, is used to refer to a sodium salt of a hemi succinate of .alpha.-dihydroqinghaosu. .alpha. and .beta. epimers of derivatives may take different physical forms when isolated in pure form, for example arteether (.beta.-epimer) is reported to be highly crystalline, while the .alpha. ethyl ether is reported to be an oil. These differences in physical properties may result in one of the epimers being more easily separated and purified. .beta.- and .alpha.-epimers of derivatives may vary in activity, for example it has been suggested in EPO patent application 330,520 that the .alpha.-ethyl ether of DQHS is inactive, however it is reported in Brossi et al. (1988 J. Med Chem. 31:645-650) that both the .alpha.-ethyl ether and arteether are potent antimalarials.
The mechanism of action of QHS and its derivatives against malaria has not been determined; however, QHS is reported to affect the trophozoite form of the malaria parasite. QHS is also reported to affect polyamine metabolism in the malaria parasite (Geary, et al. (1989) 40:240-244). It has been suggested that QHS acts to inhibit the malaria parasite by generation of active oxygen.
Klayman (1985 Science 228:1049-1055) reports that QHS, artemether, and artesunate are particularly useful for the treatment of patients with cerebral malaria, an advanced form of Plasmodium falciparum malaria that can occur when greater than 5% of erythrocytes are infected with parasites. Subsequently, it has been reported that artesunate is sensitive to hydrolysis, making it unclear as to whether the pharmacological effects are due to the parent drug or its hydrolysis product DHQHS (Brossi, et al. (1988) J. Med. Chem 31:645-650). Shwe et al. (1989) Trans. of the Roy. Soc. of Trop. Med. and Hyg. 83:489, report successful treatment of 13 cerebral malaria patients with artemether in combination with another drug.
As antimalarials, dihydroqinghaosu and artemether were reported to be one hundred times more inhibitory to P. falciparum than QHS in vitro (Li, et al. (1983) Trans. Royal Soc. Trop. Med. Hygiene 77:522-523) and in vivo in mice (China Cooperative Research Group on Qinghaosu and its derivatives as Antimalarials (1982) and Gu, et al. (1981) Journal of Chinese Medicine 2:17-24; Chung-kuo Yao Li Hsueh Pao 2(2):138-144; Chem. Abstr. 1981, 95:161913b). In mice, artemether was only four times more effective than QHS against P. berghei and P. cynomolgia. Artemether was reported to be more toxic in mice than QHS (China Cooperative Research Group on Qinghaosu and its derivatives as Antimalarials (1982) Journal of Traditional Chinese Medicine 2:31-38).
Recently, Brossi et al. (1988) J. Med. Chem. 31:645-650 reported arteether and its alpha isomer (alpha-DHQHS ethyl ether) to be potent antimalarials in vitro and in vivo (mice), equipotent with artemether and twice as potent as the natural drug QHS.
Qinghaosu and/or certain of its derivatives have also been reported to be effective in vivo (mice) against the worm Schistosoma mansoni, in vivo (rats) against the parasite Clonorchis sinensis (Klayman (1985) Science 228:1049-1055), and in vitro against Naegleria fowleri, causative agent of primary amebic meningoencephalitis (Cooke et al. (1987) J. Parasit. 73(2):411-413).
In addition, Qinghaosu and certain of its derivatives have been reported variously to be useful for the treatment of systemic lupus erythematosus (Zhuang (1979) J. New Medicine, 6:39), to have virustatic effect on influenza virus and to have an adjuvant effect on cell-mediated immunity as well as an immuno-suppressive effect (Qian et al. (1982) J. Trad. Chin. Med. 2:271).
U.S. Pat. No. 4,816,478 (issued Mar. 28, 1989) of C. R. Thornfeldt claims the use of compositions containing artelinic acid, artemether, artesunate, propyl carbonate dihydroartemisinin, or dihydroartemisinin for the treatment of AIDS or ARC (AIDS related complex).
Chinese patent application No. 85100978 (published Aug. 14, 1986 in Chinese) of Tu et al. is entitled "Reduced artemisinin for malaria and pulmonary trematodiasis treatment." The English abstract of the application suggests that it describes further applications to malaria and the use of reduced artemisinin against lung infection by the trematode Paragonimiasis.
The unique structure of QHS (i.e., the presence of a peroxide linkage) has led researchers to test the activity of endoperoxides and 1,2,4-trioxanes against the malarial parasite. Kepler et al. (1987) Journal of Medicinal Chemistry, 30(8):1505-1509, evaluated several cyclic peroxides for their antimalarial properties. The most active compound synthesized had an IC.sub.50 of 100 and 57 ng/ml, respectively, for susceptible and resistant strains of P. falciparum, as compared to an IC.sub.50 of less than 3.4 ng/ml against both forms for QHS. The compounds were all inactive in tests for blood schizonticidal activity against P. berghei. These compounds were also found to be relatively unstable at ambient temperature. The authors concluded that the peroxide linkage alone is insufficient for antimalarial activity.
Kepler et al. (1988) J. Med. Chem. 31:713-716 synthesized numerous compounds containing the 1,2,4-trioxane ring and tested their antimalarial activity. The authors found that the most active 1,2,4-trioxanes had only the same magnitude of activity as the most active endoperoxides (see Kepler, 1987, supra). These results led the authors to conclude that the 1,2,4-trioxane ring alone is not sufficient for antimalarial activity. Likewise, Jefford et al. (1988) Helvetica Chimica Acta 71:1805-1812, in evaluating the results of the antimalarial activity of their synthesized 1,2,4-trioxanes, concluded that the 1,2,4-trioxane ring itself is necessary, but not a sufficient condition to ensure significant activity. Jefford (EPO Publication No. 286,316, published Oct. 12, 1988 ) , however, claims certain synthetic 1,2,4-trioxane derivatives for use in the treatment of tropical diseases such as malaria.
Although the malarial parasite and the toxoplasmosis parasite are both protozoans of the order Eucoccidiorida, the life cycles of these organisms are significantly different. The differences in life cycle between Toxoplasma and Plasmodium are such that a drug that targets a specific stage in one parasite will not necessarily work against the other parasite. Qinghaosu is one such drug that is reported to be specific for the erythrocytic stage of Plasmodium, but has no effect on the liver (exoerythrocytic) stage. One would not expect QHS to work against Toxoplasma because the QHS susceptible life cycle stage in Plasmodium is lacking in Toxoplasma. The presence of a comparable stage of the life cycle is more important than phylogenetic proximity to provide sufficient cause to assume susceptibility to the same compound.
Chang and Pechere (1988) Transactions of the Royal Society of Tropical Medicine and Hygiene 82:867 tested the anti-Toxoplasma activity of arteether. Preliminary results suggested that arteether had some inhibitory effect on Toxoplasma replication at levels as low as 0.1 .mu.g/ml in in vitro assays using unelicited mouse peritoneal macrophages. However, the authors stated that these results were not reproducible. They further report that arteether had no inhibitory effect on the incorporation of labelled uracil by intracellular T. qondii up to concentrations of 400 .mu.g/ml. In addition, subcutaneous administration of arteether to mice infected with T. gondii was reported to be ineffective therapy for toxoplasmosis. Daily doses of 200 mg/kg were noted to increase survival time significantly, but that the treated mice eventually died of toxoplasmosis. It is also noted that arteether levels of 400 and 600 mg/kg were toxic to the animals treated. The authors concluded that arteether would not be useful in the treatment of toxoplasmosis.
Three synthetic 1,2,4-trioxanes (pentatroxane, thiahexatroxane, and hexatroxanone) have been tested for their in vitro activity against T. gondii (Chang et al. (1989) Antimicrobial Agents and Chemotherapy 33(10):1748-1752). The activities of the synthetic 1,2,4-trioxanes against intracellular T. gondii were assessed by [.sup.3 H] uracil incorporation. All three 1,2,4-trioxanes inhibited [.sup.3 H] uracil incorporation by infected macrophages. They were as effective as pyrimethamine and pyrimethamine in combination with sulfadiazine in inhibiting intracellular T. gondii. When their ability to reinfect macrophages was tested, however, none of the trioxanes affected the viability of extracellular T. gondii. Thus, the synthetic 1,2,4-trioxanes were able to block the nucleotide synthesis of the intracellular parasites, but were unable to affect viability of extracellular T. gondii.